fecal sample
Fecal samples were collected from 4 group mice upon defecation at 15th week and were stored at -80. The genomic DNA was extracted using the E.Z.N.A. stool DNA Kit (Omega Bio-tek, Norcross, GA, U.S.) according to the manufacturers protocols. V3-V4 variable regions of 16S rRNA genes was amplified by PCR (94  for 5 min, followed by 26 cycles at 94  for 30 s, 56  for 30 s, and 72  for 20 s; and a final extension at 72  for 5 min) with universal primers 343 F: TACGGRAGGCAGCAG; 798 R: AGGGTATCTAATCCT. Amplicon quality was visualized using gel electrophoresis, purified with AMPure XP beads (Agencourt), and amplified for another round of PCR. After purified with the AMPure XP beads again, the final amplicon was quantified using Qubit dsDNA assay kit. Purified amplicons were pooled in equivalent molar and paired-end sequences (2  250) on an Illumina platform according to the standard protocols.